The present invention relates to DNA sequences that contain the coding region of ammonium transporters, whose introduction into a plant genome modifies the uptake and transfer of nitrogen compounds in transgenic plants. As well as to plasmids, bacteria, yeasts, plant cells and plants containing these DNA sequences, and also to a process for the identification and isolation of DNA sequences that code for an ammonium transporter.
The supplying of growing plants with nitrogen compounds is a limiting factor in biomass production and is thus a limit on the yield of agricultural production. For this reason, nitrogen compounds, often in the form of mineral fertilisers, are added in agricultural biomass production.
An only partial uptake by the plants of the added nitrogen compounds makes it, on the one hand, necessary that the nitrogen fertiliser produced with high energy input is used in an excess, and, on the other hand, leads only to a partial uptake so that the nitrogen compounds are washed into the ground water which can lead to considerable ecological problems.
There is thus a great interest in plants which are capable of taking up large amounts of nitrogen as well as in the provision of the possibility of modifying the nitrogen uptake in plants.
For many plants there is provided information that the uptake of nitrogen is essentially in the form of nitrate salts. In strongly acid soils or in soils which follow intensive cultivation or have a strong tannin content, the nitrate formation (nitrification) is however strongly reduced and the uptake of nitrogen in the form of ammonium becomes the most important mechanism for the uptake of nitrogen compounds (Raven and Smith 1976 New Phytol 76: 415-431). Plants which are well adapted to acid soils, appear in part to favour ammonium rather than nitrate uptake and can tolerate ammonium ion concentrations that would be toxic for other plants. Examples of these plants are sugar cane, Betula verucosa or Lolium rigidum (Foy et al 1978, Ann Rev Plant Physiol 29: 511-566). The toxicity of the ammonium follows from a displacement of the ion balance: the uptake of the positively charged ammonium ion leads to an acidification of the cytoplasm, provided that no cations are secreted in counter-exchange. In an uptake of ammonium via a transport system whose uptake mechanism is based on an ammonium ion proton antiport, the ionic imbalance is not a problem.
There is thus a great interest in transport systems which work by the mechanism of an ammonium ion proton antiport and/or in systems which could be converted through techniques of protein engineering into ammonium ion proton antiports.
In spite of extensive efforts, it has not been possible up until now to isolate transport systems with whose help plants are protected against an ammonium ion loss caused by membrane diffusion (retrieval system).
By the term ammonium is also to be understood methylamine which is analogous to ammonium.
An active uptake system has been investigated in the fungus Aspergillus nidulans (e.g Arst et al., 1973, Mol Gen Genet 121: 239-245), as well as in Penicillum chrysogenum (Hackefte et al., J Biol Chem 245: 4241-4250). In these studies methylamine was used as the ammonium analogue. For Aspergillus nidulans, five genetic loci were established, which take part in the transport of methylamine (Pateman et al., 1973, J Bacteriol 114: 943-950). In biochemical experiments concerning the methylammonium transport in Penicillum chrysogenum and Saccharomyces cerevisisae, it has been shown that the transport is temperature and pH dependent, and that the pH optimum is 6.0 to 6.5 and the transport efficiency steadily rises up to a temperature of 35xc2x0 C. (Roon et al., 1975, J Bacteriol 122: 502-509). The methylamine and/or ammonium transport in Saccharomyces cerevisiae is dependent on the supply of easily usable chemical energy, e.g. in form of glucose. The transport system consists of at least three independent transporters, which differ in transport capacity and affinity for the substrate: besides a high affinity transporter with low capacity (Km value=250 xcexcM, maximum speed Vmax=20 nmol/min per mg cells (dry weight)) there is a low affinity system with high capacity (Km=2 mM, Vmax=50 nmol/min per mg cells) and a low affinity system with medium capacity (Km=20 mM, Vmax=33 nmol/min per mg cells) (Dubois and Grenson, 1979, Mol Gen Genet 175: 67-76). In the presence of glutamine or asparagine in the surrounding medium, the ammonium uptake is reduced by 60 to 70%, while other amines hardly have any influence (Roon et al., 1975, J Bacteriol 122: 502-509).
Nothing is known about the molecular nature of the ammonium transporter of the above-mentioned or different fungi and other organisms, such as for example bacteria. Equally nothing is known about systems, with whose help fungi or bacteria protect against ammonium ion loss by membrane diffusion (retrieval systems) at the molecular level. Further genes, which code for ammonium transporters are not known.
Various evidence suggests that the ammonium transport in Saccharomyces cerevisiae is accomplished by at least two functionally different transport systems (Dubois and Grenson, 1979, Mol Gen Genet 175: 67-76):
1. Kinetic analyses of the methylamine-uptake by Saccharomyces show an abrupt transition between apparent linear sections, whereby both functions are inhibitable by ammonium.
2. Both functions can be separately excluded by mutation. The resulting mutations mep-1 and mep-2 are genetically independent.
3. The mutants mep-1 and mep-2 can each grow in media with ammonium as the only nitrogen source, while a double mutant mep-1/mep-2 shows hardly any growth under these conditions (data taken from Dubois and Grenson, 1979).
A clarification of the ammonium transport processes in plants, that leads to similar detailed information such as for yeast, is not available and because of the difficulty of the molecular biological analysis of mutations is scarcely possible in a corresponding manner.
The object of the present invention is to provide coding for DNA-sequences of ammonium transporters which cause a change in the uptake and transfer of nitrogen compounds in transgenic plants.
The object of the present invention is further to provide DNA-constructs, such as plasmids, with which the ammonium transport in transgenic plants can be modified by introduction of the corresponding construct (plasmids) into the plant genome which leads upon transcription to the formation of a new ammonium transporter molecule in the transgenic plant and/or the suppression of the formation the plant""s own ammonium transporter molecules.
There have now surprisingly been found DNA sequences, that contain the coding region of a plant ammonium transporter, whereby the information contained in the nucleotide sequence when integrated in a plant genome
a) under the control of a promoter in a sense orientation makes the expression of a translatable mRNA which leads to the synthesis of an ammonium transporter in transgenic plants possible or
b) under the control of a promoter in an anti-sense orientation makes the expression of a non-translatable mRNA which prevents the synthesis of an endogenous ammonium transporter in transgenic plants possible.
A further aspect of the invention is to provide DNA sequences which contain the coding region of a plant ammonium transporter.
In an analogous way, the ammonium transporter can also be used to modify animal cells.
The DNA sequences, which code for a plant ammonium transporter, can be identified and isolated by a process of determining which DNA sequences are able to complement specific mutations in the yeast Saccharomyces cerevisiae. In general, the mutations, are those which have the result that the corresponding strains cannot grow any further in media which contain ammonium as the only nitrogen source. Such a strain can be transformed with a plant cDNA-library and transformands can be selected which can grow in media which contain ammonium as the only nitrogen source.
A further aspect of the invention is a process for the identification and isolation of DNA sequences that code for ammonium transporters from plants, which includes the following steps:
a) transformation of a yeast strain which cannot grow in media which contains ammonium as the only nitrogen source with a plant cDNA-library using suitable expression vectors,
b) selection and propagation of transformants, which after expression of plant cDNA-sequences, can grow in media which contain ammonium as the only nitrogen source, and
c) isolation of the expression vectors which carry a plant cDNA insert from the selected transformand.
The yeast strain in process step a) is preferably one which cannot take up ammonium from the medium because of mutations in the transport systems for the ammonium uptake. Preferred is the double mutant mep1/mep2 (strain 26972c), described by Dubois and Grenson (1979, Mol. Gen. Genet. 175:67-76), and with which two uptake systems for ammonium are interrupted following mutation.
A further aspect of the invention is to provide DNA sequences from plants which are obtainable using the above described process and which code for a protein with the biological activity of an ammonium transporter.
Whether it is possible using such complementation process to identify DNA sequences which code for plant ammonium transporters depends on various factors. First the expression plasmid suitable for use in yeast must contain cDNA fragments which code for the plant ammonium transporter, that means the mRNA fundamental for the cDNA synthesis must result from tissues which express the ammonium transporter. Since nothing is known about plant ammonium transporters, tissues which code for the ammonium transporter are therefore also not know.
A further prerequisite for the success of the complementation strategy is that ammonium transport systems existing in plants can be functionally expressed in yeast, since only in this case is a complementation of the deficiency liberated by the mutation possible. Since nothing is known about plant ammonium transporters, it is also not known whether a functional expression in yeast is possible.
It has now further been surprisingly found that by expression of a cDNA library, for example from leaf tissue of Arabidopsis thaliana, by means of expression plasmids suitable for use in yeast which contain the promoter of phosphoglycerate kinase from yeast, the complementation of the double mutation mep-1/mep-2 is possible, if the expression plasmids contain specified plant cDNA fragments. These cDNA fragments code for plant ammonium transporters.
The identification of plant ammonium transporters is described here using Arabidopsis thaliana as an example, but it is not however limited to this plant species.
A cDNA fragment that codes for a plant ammonium transporter containing for example the following sequence.
(Seq. ID No.1):
The DNA sequences of the invention, identified using the transformed yeast strain, such as e.g. the sequence Seq. ID No. 1, can be introduced into plasmids and thereby be combined with regulatory elements for expression in eukaryotic cells (see Example 4). These regulatory elements are, on the one hand, transcription promoters, and, on the other hand, transcription terminators. With the plasmids, eukaryotic cells can be transformed, with the aim of expression of a translatable mRNA which makes possible the synthesis of an ammonium transporter in the cells (sense orientation) or with the aim of expression of a non-translatable RNA which prevents the synthesis of an endogenous ammonium transporter in the cells (anti-sense orientation).
These plasmids are also an aspect of the invention.
Preferred plasmids are plasmids p35S-MEP-a and p35S-a-MEP-a, which have been deposited at the Deutsche Sammlung von Mikroorganismen (DSM).
A still further aspect of the present invention are yeasts which contain the DNA sequences of the invention.
The yeast strains used for the identification of a plant methylamine or ammonium transporter can be used to study the properties of the transporter as well as its substrate. The DNA sequences of a plant ammonium transporter present in plasmids can, in accordance with results of these studies, be subjected to a mutagenesis or sequence modification by recombination in order to change the properties of the transporter. By changing the specificity of the transport system, the transport of new compounds is possible, which opens up interesting applications (see below). In addition, by suitable change of the transporter, the transport mechanism can be modified. One can envisage, especially but not exclusively, a change, that modifies the cotransport properties of the transporter, for example with the effect that this results in an ammonium ion-proton-antiport, which prevents a toxicity of ammonium ions taken up for plants as a result of acidification of the cytoplasm. In this way, the yeast strain of the invention, which contains the cDNA sequence of a plant ammonium transporter in an expression plasmid, can be used for a mutation selection system.
By expression of an RNA, corresponding to the DNA sequences of the invention, of plant ammonium transporters in transgenic plants it is possible to have a change of the plant nitrogen metabolism, whose economic significance is obvious. Nitrogen is the nutrient mainly responsible for limiting growth. The viability of seedlings as well as germination capacity of seeds is directly dependent on the nitrogen content of storage tissue. The formation of high value food materials with a high protein content is dependent on a sufficient nitrogen supply.
The change of nitrogen uptake, for example by addition of a new uptake system of nitrogen compounds such as ammonium, can thus lead to an increase in yield of transgenic crops, especially under nitrogen limitation. In this way, transgenic plants can be cultivated in high yields under low input conditions.
The possibility of suppressing the uptake of ammonium in the transgenic plants can however also be desirable under certain conditions. For example, one can envision the cultivation of transgenic plants on acid soils which are not suitable for growth. As a result of suppressing nitrification, ammonium ion concentrations on such soils could be present which would be toxic for certain plants. This is because the ammonium taken by the plants in the cells can no longer be fully metabolised and acts as a cell poison. The suppression of the uptake of ammonium by suppressing the biosynthesis of the ammonium transporter can thus alter transgenic plants to the extent that cultivation on acid soils becomes possible.
A further aspect of the present invention are transgenic plants in which the DNA sequences of the invention are introduced as a constituent of a recombinant DNA molecule, in which this recombinant DNA molecule is stably integrated into the genome, and in whose cells based on the presence of these sequences there is achieved either a synthesis of an additional plant ammonium transporter whereby these cells can take up larger amounts of ammonium in comparison with untransformed plants or there is achieved an inhibition of the synthesis of endogenous ammonium transporters whereby such cells show a reduced uptake of ammonium in comparison with untransformed cells.
Transgenic crops are, for example tobacco, potatoes, sugar beet, soya beans, peas, beans or maize.
The genetic modification of dicotyledonous and monocotyledonous plants can be carried out by currently known processes, (see for example Gasser, C. S., Fraley, R. T., 1989, Science 244:1293-1299; Potrykus, 1991, Ann Rev Plant Mol Biol Plant Physiol 42: 205-225). For expression in plants the coding sequences must be coupled with the transcriptional regulatory elements. Such elements called promoters, are known (see for example EP 375091).
Further, the coding regions must be provided with transcription termination signals with which they can be correctly transcribed. Such elements are also described (see Gielen et al., 1989, EMBO J 8: 23-29). The transcriptional start region can be both native and/or homologous as well as foreign and/or heterologous to the host plant. If desired, termination regions are interchangeable with one another. The DNA sequence of the transcription starting and termination regions can be prepared synthetically or obtained naturally, or obtained from a mixture of synthetic and natural DNA constituents. For the introduction of foreign genes into higher plants, a large number of cloning vectors are available that include a replication signal for E. coli and a marker which allows a selection of the transformed cells. Examples of such vectors are pBR 322, pUC-Series, M13 mp-Series, pACYC 184 etc. Depending on the method of introduction of the desired gene into the plants, other DNA sequences may be suitable. Should the Ti- or Ri-plasmid be used, e.g. for the transformation of the plant cell, then at least the right boundary, and often both the right and left boundary of the Ti- and Ri-Plasmid T-DNA, is attached as a flanking region to the gene being introduced. The use of T-DNA for the transformation of plants cells has been intensively researched and is well described in EP 120 516; Hoekama, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V. Alblasserdam, (1985), Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46 and An et al. (1985) EMBO J. 4: 277-287. Once the introduced DNA is integrated in the genome, it is generally stable there and remains also in the offspring of the original transformed cells. It normally contains a selection marker, which induces resistance in the transformed plant cells against a biocide or antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc. The individual marker employed should therefore allow the selection of transformed cells from cells which lack the introduced DNA.
For the introduction of DNA into a plant host cell, besides transformation using Agrobacteria, there are many other techniques available. These techniques include the fusion of protoplasts, microinjection of DNA and electroporation, as well as ballistic methods and virus infection. From the transformed plant material, whole plants can be regenerated in a suitable medium, which contains antibiotics or biocides for selection. The resulting plants can then be tested for the presence of introduced DNA.
A further aspect of the present invention is to provide transformed plant cells in which the DNA sequences of the invention are introduced as a constituent of a recombinant DNA molecule in which the recombinant DNA molecule is stably integrated into the genome. In the cells, based on the presence of these sequences, there is achieved either a synthesis of an additional plant ammonium transporter whereby the cells can take up larger amounts of ammonium in comparison with untransformed plants or there is achieved an inhibition of the synthesis of endogenous ammonium transporters whereby such cells show a reduced uptake of ammonium in comparison with untransformed cells.
No special demands are placed on the plasmids in injection and electroporation. Simple plasmids, such as e.g. pUC-derivatives can be used. Should however whole plants be regenerated from such transformed cells the presence of a selectable marker gene is necessary. The transformed cells grow within the plants in the usual manner (see also McCormick et al. (1986) Plant Cell Reports 5: 81-84). These plants can be grown normally and crossed with plants, that possess the same transformed genes or different genes. The resulting hybrid individuals have the corresponding phenotypical properties.
The introduction of DNA plant ammonium transporters for changing the uptake of nitrogen compounds is described here using Arabidopsis thaliana and tobacco as examples. The use is not however limited to this plant species. The DNA sequences of the invention can also be introduced in plasmids and thereby combined with steering elements for an expression in prokaryotic cells. The formation of a translatable RNA sequence of a eukaryotic ammonium transporter from bacteria leads, in spite of the considerable differences in the membrane structures of prokaryotes and eukaryotes, to the expression in prokaryotes of a functional eukaryotic ammonium transporter with its substrate specificity. This makes possible the production of bacterial strains which, as for the yeast strain used for identifying the ammonium transporter, could be used for studies of the properties of the transporter as well as its substrate, which opens up interesting applications.
The invention also relates to bacteria that contain the plasmids of the invention.
The DNA sequences of the invention can also be introduced in plasmids which allow mutagenesis or a sequence modification through recombination of DNA sequences using standard microbiological processes. In this way the specificity of the ammonium transporter can be modified.
Modified ammonium transporters can be used for example for the transformation of agriculturally useful transgenic plants, whereby both transport of pesticides and plant growth regulators to plants can be envisaged.
By using standard processes (see Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2. Edn., Cold Spring Harbor Laboratory Press, N.Y., USA), base exchanges can be carried out or natural or synthetic sequences can be added. In order to fuse DNA fragments with one another, adaptors or linkers can be added to the fragments. Further, manipulations can be carried out which prepare suitable restriction cleavage sides or to remove the excess DNA or restriction cleavage sites. Where insertions, deletions or substitutions such as, for example, transitions and transversions are to be carried out, in vitro mutagenesis, primer repair, restrictions or ligations can be used. For methods of analysis, in general, a sequence analysis, restriction analysis and other biochemical molecular biological methods can be used. After each manipulation the DNA sequence used, can be cleaved and fused with another DNA sequence. Each plasmid sequence can be cloned in the same or different plasmids.
The invention also relates to derivatives or parts of plasmids on which the DNA sequences of the invention are localised.
Derivatives or parts of the DNA sequences and plasmids of the invention can also be used for the transformation of prokaryotic and eukaryotic cells.
Further, the DNA sequences of the invention can be used according to standard processes for the isolation of homologous sequences from the genome of plants of various species, which also code for ammonium transporter molecules. With these sequences, constructs for the transformation of plant cells can be prepared which modify the transport processes in transgenic plants.
By the terms xe2x80x9chomologyxe2x80x9d or xe2x80x9chomologous sequencesxe2x80x9d are to be understood, a sequence identity of 60% to 80%, preferably 80% to 95% and especially 95% to 100%.
In order to specify related DNA sequences, gene libraries must first be prepared which are representative of the content of genes of a plant species or of the expression of genes in a plant species. The former are genomic libraries, while the latter are cDNA libraries. From these, related sequences can be isolated using the DNA sequences of the invention as probes. Once the related gene has been identified and isolated, a determination of the sequence and an analysis of the properties of the proteins coded from this sequence is possible.
DNA sequences of ammonium transporters obtained in this way are also part of the invention and could be used as described above.
The use of the DNA sequences as described above is also part of the invention.
A further aspect of the invention are DNA sequences from plants, which hybridise with DNA sequences of the invention and code for a protein that possesses the biological activity of an ammonium transporter. The term xe2x80x9chybridisationxe2x80x9d means in this connection, a hybridisation under conventional hybridisation conditions, preferably under stringent conditions, such as described for example by Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, 2. Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, N.Y.). An important biological activity of an ammonium transporter is the capability of transporting ammonium or analogues thereof through biological membranes. This activity can be measured by uptake of ammonium or analogues thereof by cells which express the particular ammonium transporter, as described for example in example 3 of the present invention.
Deposits
The following plasmids were deposited at the Deutschen Sammlung von Mikroorganismen (DSM) in Braunschweig, Germany on the 26.10.1993 (deposit number):